Domain swapping oligomerization of thermostable c-type cytochrome in E. coli cells

Knowledge on domain swapping in vitro is increasing, but domain swapping may not occur regularly in vivo, and its information in cells is limited. Herein, we show that domain-swapped oligomers of a thermostable c-type cytochrome, Hydrogenobacter thermophilus cyt c(552), are formed in E. coli which expresses cyt c(552). The region containing the N-terminal α-helix and heme was domain-swapped between protomers in the dimer formed in E. coli. The amount of cyt c(552) oligomers increased in E. coli as the cyt c(552) concentration was increased, whereas that of high-order oligomers decreased in the order of decrease in protein stability, indicating that domain swapping decreases in cells when the protein stability decreases. Apo cyt c(552) was detected in the cyt c(552) oligomer formed in E. coli, but not in that of the A5F/M11V/Y32F/Y41E/I76V mutant. The cyt c(552) oligomer containing its apo protein may form at the periplasm, since the apo protein detected by mass measurements did not contain the signal peptide. These results show that domain-swapped cyt c(552) oligomers were formed in E. coli, owing to the stability of the transient oligomer containing the apo protein before heme attachment. This is an indication that exceedingly stable proteins may have disadvantages forming domain-swapped oligomers in cells.